The importance of polyunsaturated fatty acids (or “PUFAs”) are undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA; 18:2) or α-linolenic acid (ALA; 18:3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or TAGs; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of ω-3 PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) produce cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al., Lancet 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., and Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Furthermore, numerous other studies document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 fatty acids against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
Based on the tremendous scientific knowledge in support of the benefits of a diet comprising long-chain PUFAs for humans and other animals, considerable research has been directed toward the understanding and discovery of genes encoding the biosynthetic pathways that permit synthesis of lipids and fatty acids. As a result, numerous studies have attempted to introduce pathways that enable ω-3/ω-6 PUFA biosynthesis into organisms that do not natively produce (ω-3/ω-6 PUFAs as a preliminary means to demonstrate the feasibility of this approach. One such organism that has been extensively manipulated is the non-oleaginous yeast, Saccharomyces cerevisiae. Specifically, Dyer, J. M. et al. (Appl. Envi. Microbiol., 59:224-230 (2002)) reported synthesis of ALA upon expression of two plant fatty acid desaturases (FAD2 and FAD3); Knutzon et al. (U.S. Pat. No. 6,136,574) expressed one desaturase from Brassica napus and two desaturases from the fungus Mortierella alpina in S. cerevisiae, leading to the production of LA, γ-linolenic acid (GLA), ALA and stearidonic acid (STA); and Domergue, F. et al. (Eur. J. Biochem. 269:4105-4113 (2002)) expressed two desaturases from the marine diatom Phaeodactylum tricornutum in S. cerevisiae, leading to the production of EPA (0.23% with respect to total fatty acids). However, none of these preliminary results are suitable for commercial exploitation.
Other efforts to produce large-scale quantities of ω-3/(ω-6 PUFAs have relied on the cultivation of microbial organisms that natively produce the fatty acid of choice [e.g., EPA is produced via: heterotrophic diatoms Cyclotella sp. and Nitzschia sp. (U.S. Pat. No. 5,244,921); Pseudomonas, Alteromonas or Shewanella species (U.S. Pat. No. 5,246,841); filamentous fungi of the genus Pythium (U.S. Pat. No. 5,246,842); or Mortierella elongata, M. exigua or M. hygrophila (U.S. Pat. No. 5,401,646)]. However, these methods all suffer from an inability to substantially improve the yield of oil or to control the characteristics of the oil composition produced, since the fermentations rely on the natural abilities of the microbes themselves. Furthermore, large-scale fermentation of some organisms (e.g., Porphyridium, Mortierella) can also be expensive and/or difficult to cultivate on a commercial scale.
A recent alternative to the strategies above is that of Picataggio et al. (see WO 2004/101757 and co-pending U.S. patent application Ser. No. 60/624812, each herein incorporated entirely by reference), wherein the utility of the oleaginous yeast Yarrowia lipolytica (formerly classified as Candida lipolytica) has been explored as a preferred class of microorganisms for production of ω-3/(ω-6 PUFAs such as arachidonic acid (ARA), EPA and DHA. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation can be up to about 80% of the cellular dry weight. Despite a natural deficiency in the production of ω-6 and ω-3 fatty acids in these organisms (since naturally produced PUFAs are limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids)), Picataggio et al. (supra) have demonstrated production of up to 28% EPA (of total fatty acids) by introduction of PUFA desaturases, elongases and acyltransferases. Despite this success, a general method of up-regulating ω-3/ω-6 fatty acids synthesis and accumulation in the lipid and oil fractions has not been previously taught.
With respect to plants, annual and perennial oilseed crops produce a yearly output of greater than 87 million tonnes in traded vegetable oils that is worth about US $45-50 billion (Murphy, D. J. Appl. Biotech, Food Sci. and Policy, 1(1):25-38 (2003)). Although many modifications could improve the edible quality of plant oils, the introduction of long-chain (ω-3 PUFAs is one of the top two targets for those working in agricultural biotechnology (since mosses and algae are the only known plant systems that produce considerable amounts of ω-3 PUFAs such as EPA and DHA). As such, seed oil content and composition has been manipulated by introduction of PUFA desaturases, elongases and acyltransferases into several well-studied oilseed crop plants (e.g., flax, rape, soybean [as described in e.g., WO 2003/093482; WO 2004/057001; WO 2004/090123; WO 2004/087902; U.S. Pat. Nos. 6,140,486; 6,459,018; U.S. 2003/0172399; U.S. 2004/0172682; U.S. 2004/098762; U.S. 2004/0111763; Qi, B. et al., Nature Biotech. 22:739-745 (2004); Abbadi et al., The Plant Cell, 16:2734-2748 (2004)]). The greatest accumulation of EPA in these studies is 19.6% of total fatty acids in transformant soybean seeds, by expression of various PUFA desaturases and elongases (U.S. 2004/0172682). However, despite the extensive work described above, none of these studies have set forth a means to increase the percent of PUFAs in the total lipid and oil by regulation of the host organism's native acyltransferases.
Acyltransferases are intimately involved in the process of triacylglycerol (TAG) biosynthesis, wherein newly synthesized PUFAs are transferred into a host organism's storage lipid pools. This is possible since most free fatty acids become esterified to coenzyme A (CoA) to yield acyl-CoAs. These molecules are then substrates for glycerolipid synthesis in the endoplasmic reticulum of the cell, where phosphatidic acid and 1,2-diacylglycerol (DAG) are produced. Either of these metabolic intermediates may be directed to membrane phospholipids or DAG may be converted to TAG by the addition of a fatty acid to the sn-3 position of DAG. This reaction is catalyzed by a diacylglycerol acyltransferase enzyme (DAG AT), such as a diacylglycerol acyltransferase 1 (DGAT1), diacylglycerol acyltransferase 2 (DGAT2) or a phospholipid:diacylglycerol acyltransferase (PDAT).
In the present disclosure, the Applicants describe methods to regulate the percent of PUFAs within the lipids and oils of PUFA-producing oleaginous organisms, by regulating the activity of a host organism's native DAG ATs. Specifically, since oil biosynthesis is expected to compete with polyunsaturation during oleaginy, it is possible to reduce or inactivate the activity of an organism's one or more DAG ATs (e.g., PDAT and/or DGAT1 and/or DGAT2), to thereby reduce the overall rate of oil biosynthesis while concomitantly increasing the percent of PUFAs that are incorporated into the lipid and oil fractions.
Thus, the Applicants have solved the stated problem wherein methods to increase the percent of PUFAs in the total lipid and oil fractions of oleaginous organisms were previously lacking, by enabling one to engineer a wide variety of oleaginous organisms (e.g., bacteria, algae, moss, yeast, fungi, plants) to produce lipids and oils with very specific fatty acid compositions using techniques that rely on manipulation of the host organism's native DAG ATs.